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LAB 2
Bacterial Staining Techniques I
I. Complete Lab 1
II. Staining Microorganisms: Simple Stains (Direct and Negative)
III. Morphological Unknown
I. Complete Lab 1:
Collect your plates from the trays on the side bench. Observe the TSA plates for colonies of various
sizes, shapes and colors. Each bacterial or fungal species gives a characteristic colony color and
morphology. Draw the colonies observed on both TSA plates in the spaces provided in the Results
section of Lab #1. Pick three colonies from either of the TSA plates and describe the colony color and
morphology. Also observe the cloudiness (turbidity) of your nutrient broth tube and estimate the number
of bacteria per mL (see turbidity table below).
TERMS AND DEFINITIONS
Colony: a single cell divides exponentially forming a small, visible collection of cells. Colonies are
observed when bacteria are grown on a solid medium. Each colony usually contains 107-108 bacteria.
Colony morphology: Characteristics of a colony such as shape, edge, elevation, color and texture.
Turbidity: cloudy appearance of a liquid medium due to the presence of bacteria. You can "estimate"
the number of bacteria per mL by using the table below.
Turbidity # Bacteria per mL
none 0 - 106
light 107
moderate 108
*heavy 109
*Usually bacterial populations do not exceed 3 x 109 bacteria/mL when grown in liquid media.
II. STAINING MICROORGANISMS
A. Smear preparation
Simple Stains:
B. Direct stain
C. Negative stain
TERMS AND DEFINITIONS
Chromophores: Groups with conjugated double bonds that give the dye its color.
Direct, cationic, basic or positive dyes: contain positively charged groups. Examples include methylene
blue, basic fuchsin, and crystal violet. These dyes directly bind to and stain the negatively charged
surface of bacterial cells.
Negative, anionic, or acidic dyes: contain functional groups that have a negative charge. Examples
include eosin, nigrosin and Congo red. These dyes are repelled by the negatively charged surface of
bacterial cells. Thus, they stain the background, leaving the bacterial cells clear and bright against a dark
background.
Heat Fixation: application of heat to a bacterial smear preparation. This procedure simultaneously kills
and attaches the bacteria to the slide.
